Molecular cloning and characterization of two Arabidopsis thaliana sulfotransferases

Abstract

Two Arabidopsis thaliana sulfotransferases, AtST4a and AtST6 , were identified through BLAST searches of The Arabidopsis Information Resource's (TAIR) database using AtST1 as a query sequence. The intronless AtST4a was cloned via PCR from Arabidopsis thaliana ecotype Col-0 genomic DNA. A cDNA clone of AtST6 was obtained and fully sequenced. AtST4a and AtST6 were both cloned into the bacterial expression vector pQE30. The recombinant proteins expressed in E. coli were 42 and 40 KDa, respectively. It was determined that AtST4a displayed specificity for various stereoisomers of brassinosteroids and their immediate precursors. AtST6 was found to sulfonate a wide range of primary and secondary alcohols with unknown kinetics. Molecular characterization of AtST4a and AtST6 revealed that the deduced protein sequence of both enzymes contain all the amino acids involved in PAPS binding and catalysis (Marsolais et al. 1995). Expression studies failed to determine conditions under which the genes preferentially expressed. Putative transgenic plants constitutively expressing AtST4a in the sense orientation produced a phenotype similar to that of the known brassinosteroid deficient mutants